476 J. EUK. MICROBIOL.. VOL. 42. NO. 5. SEPTEMBER-OCTOBER 1995

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17508 1-5095.

Received 9-1-94, 2-2-95; accepted 3-27-95.

J Euk. Mlcrobrol.. 42(5). 1995. pp. 4 7 6 4 9 0 t c j 1995 by the Soctety of Prorozooloyslr

Morphology and Ecology of Sirofoxophyffum utriculariae (Penard, 1922) N. G., N. Comb.

(Ciliophora, Pleurostomatida) and an Improved Classification of Pleurostomatid Ciliates

WILHELM FOISSNER* ' and DETLEV LEIPE** *** *L~tirver.\rtur Sa1:hirr:y. lnsrrrirr tur Zoologre. Hellhrunnerstrasst.~ 34, '-1-5020 Sakhurg. Auslria

**Freir L'ni~c~rsirat Berlin, ln.stitu~./iir Zoologre. Kiinigiti-Luise-Sfrassr 1-3. 0-141 95 Berlin. Germany. and ***Nutional C'entcr.lbr Biorechnolog~ Ir~forniation, Building 38.4, Berhesda. Maryland 20894, USA

ABSTRACT. The morphology and infraciliature of Sirolo.\-opti?.llut,1 irfriculariae (Penard. 1922) n. g., n. comb. were studied in live cells. with the scanning and transmission electron mlcroscope. as well as in specimens impregnated with protargol and silver carbonate. The new genus, Sirolo.uo~~h~~llur~7. belongs to the Loxophyllidae and has a specific combination of characters, viz. an oral bulge surrounding almost the entire cell. three perioral kineties. a single brush kinety. and a single right dorsolateral kinety. The ecology and faunistics of S. utricir/ariae are reviewed. I t is a rare and infrequent predator prefemng clean freshwaters. The somatic monokinetid of S. utriculariae has typical haptorid ultrastructure. including two transverse microtubular ribbons. The oral bulge is patterned string-like with riffles containing the transverse microtubular ribbons originating from the oral kinetids. Perioral kineties I and 2 consist of dikinetids having one basal body each ciliated: the nonciliated basal body is associated with a nematodesmal and a transverse microtubular ribbon. Perioral kinety 3 consists of ciliated monohnetids having a fine structure similar to the somatic kinetids: they form triads with the dikinetids from perioral kinety 2. The classification of pleurostomatid ciliates is reviewed. Two suborders (Amphileptina, Litonotina) and three families (Amphileptidae. Litonotidae. Loxophyllidae n. fam.) are recognized and defined.

Supplementary key words. .4citwriu, Haptoria. infraciliature. Lironotus, Lo.\-ophyllurn. Opisthodon, Pseudoamphileptus, taxonomy, ultrastructure.

HE pleurostomatid ciliates have attracted comparatively T few ciliatologists. possibly because they are well circum- scribed a n d their close relationship with haptor id ciliates has

' To whom correspondcncc should be addressed.

never been questioned. However , m a n y new species have been described since the revision by Kah l [28], most by Vuxanovici [45.46] a n d Song & Wilbert [4 I] . Foissner's group studied the infraciliature of representatives of mos t known genera a n d pro- vided improved diagnoses for Amphileptus [ 131, Litonofus [ 131, Aciricria [ I ] a n d Lo.rophy//urvi [23]. Foissner [ 121 also estab-

FOISSNER & LEIPE-SIROLOXOPHYLLUM N. G. 477

lished a new genus, Pseudoaniphileptus, for Amphileptus ma- crostorna and rediscovered the long missed genus Opisthodon [13], already placed on the nomina oblita list by Corliss [S]. These data and some electron microscope studies [2, 371 pro- vided a firm base for an improved classification of the group

More recently, Lipscomb & Riordan [34] destroyed the ho- mogeneity of the pleurostomatids by including typical haptorids like Spathidium and Didinium. This view is not supported by the present results which emphasize the structural and onto- genetic peculiarities of the pleurostomatids, setting them up clearly from the haptorids s . str.

[211.

MATERIALS AND METHODS Organisms and preparations. The two populations of S. utri-

culariue studied were isolated from a slowly running stream in Berlin and from the sludge of a rapid gravity filter of the Bad Fussing waterworks near Munich [ 181. Both populations could be cultured for some time on diluted lettuce medium enriched with dried yolk to provide bacterial food for their prey, mainly Glaucoma scintillans and Cinetochilurn inargaritaceum.

Cells were studied in vivo using a high-power oil immersion objective and differential interference contrast [ 141. Protargol [ 16; protocol I] and silver carbonate [ 151 were used to reveal the infraciliature. Preparations for scanning (SEM) and trans- mission (TEM) electron microscopy were performed as de- scribed previously [ 17, 321.

Counts and measurements on silvered specimens were per- formed at a magnification of x 1.000. In vivo measurements were conducted at a magnification of x 100-1,000. Although these provide only rough estimates it is worth giving such data as specimens usually shrink in preparations or contract during fixation. The standard deviation and coefficient of variation were calculated for morphometric data. Drawings of live spec- imens are based on free-hand sketches, those of impregnated cells were made with a camera lucida.

Terminology. Standard terminology as outlined in [5 , 351 is applied for the light and electron microscope data. A few un- common terms used in the light microscope description and the generic key are explained in the following paragraphs.

Dorsal brush. Row(s) of shortened, paired cilia near the an- terior dorsal margin of the cell (Fig. 3, 7, 29). Usually, these rows are continuous with the anterior ends of one or several left lateral somatic kineties.

Dorsolateral kineties. Two kineties at the right (Loxophyllum [23]), or one kinety each at the right and left (Siroloxophyllum, Fig. 6, 19, 30) margin of the dorsal side; distinguished from regular somatic ciliary rows either by shortened cilia and/ or in extending around the posterior end of the cell, forming a more or less distinct suture with the abutting posterior ends of the regular somatic kineties.

Oral bulge. A nonciliated eminence along the oral slit, often indistinct in pleurostomatids. Appears more or less distinctly string-like patterned in SEM-micrographs (Fig. 10, 29). The actual oral opening is defined as the bulge region which is ac- companied by that portion of perioral kineties 1 and 2 which has paired basal bodies and nematodesmata.

Perioral kineties. Two or three kineties lining the oral bulge, usually continue posteriorly as somatic ciliary rows. Kineties 1 and 2 always composed of paired basal bodies, at least along oral opening (Fig. 7, 8, 1 1 , 26). Perioral kinety 1 lines the left bulge wall, kineties 2 and 3 the right [ 131.

Spica. A suture formed by shortened ciliary rows in the mid- line of the anterior right body half; typically found in Amphi- leptus (Fig. 49).

RESULTS Siroloxophyllum n. g.

Diagnosis. Loxophyllidae with oral bulge surrounding almost entire cell. Three perioral kineties extending from anterior end to mid-body. Single brush kinety near dorsal margin. Single right dorsolateral kinety.

Type species. .4mphileptus utriculuriae Penard, 1922. Etymology. Composite from the Greek words siro (string).

10x0s (oblique) and phyllunz (leaf). Neuter gender. Name refers to string-like appearance of oral bulge.

Type specimens. One holo (genus) type slide and one voucher slide of protargol impregnated Siroloxophyllum. Munich pop- ulation, have been deposited in the collection of microscope slides of the Oberosterreichische Landesmuseum in Linz (LI). Austria. Accession numbers: 26, 27/1994. The slides also serve as neotypes for the species, .4niphiIeptirs ictriculariur Penard, 1922 [36], because Song & Wilbert [41] made no mention of deposited neotype material,

Redescription of Siroloxophyllum utriculariae (Penard, 1922) n. comb.

Light and scanning electron microscopy. Morphometric data shown in Table 1 are repeated in this section only as needed for clarity. Many characters of S. utriculariae and of other pleu- rostomatids [ 13, 231 vary greatly, as indicated by the rather high coefficients of variation (most 2 15%).

Size highly variable within and between populations, in vivo 65-270 x 20-80 pm according to our observations and litera- ture data [27, 28, 36, 4 l], usually about 140-200 x 30-60 prn: up to 30% contractile, fixed and stained specimens thus smaller due to contraction and shrinkage. Shape likewise highly vari- able, often, however, lanceolate with widest portion in or close underneath mid-body, anterior half usually more distinctly nar- rowed than posterior and slightly curved dorsally, but not snout- like as in Loxophyllum; anterior end narrowly rounded, pos- terior end broadly rounded to elongated. Field material flattened leaf-like (up to 4: l), with very flat and hyaline, about 7 pm wide fringe containing extrusomes. Right side flat to slightly concave. left more or less distinctly vaulted bearing 3-8 distinct crests in central third; crests 2-5 pm high and 2 pm wide, gradually flattened toward body ends, become inconspicuous and even disappear in well-fed specimens (Fig. 1 , 4. 5 , 14. 15, 21, 22).

Nuclear apparatus in or near centre ofcell, stands out as bright blister against darker, granulated cytoplasm (Fig. 1). Usually two macronuclear nodules and one micronucleus; number con- stant in Berlin and Bonn population, highly variable in speci- mens from Munich with, however, a median of two as in the other populations (Table 1). Macronuclear nodules small as compared to size of cell, slightly ellipsoid, often close together ( 5 2 pm). micronucleus then not within but on cleft; nucleoli roundish, distributed throughout nodules (Fig. 7, 14, 15, 18). Micronucleus slightly ellipsoid, 2-3 x 2 pm, within or on cleft formed by macronuclear nodules (Fig. 1).

Two contractile vacuoles, each with numerous excretory pores on right surface (Fig. 24), in anterior and posterior third of cell, respectively; anterior vacuole near ventral side, posterior vac- uole near dorsal side, thus forming highly characteristic diagonal pattern with nuclear apparatus in between (Fig. 1, 14, 21).

Extrusomes (toxicysts) 6-8 pm long, thin (diameter about 0.4 pm) and slightly curved, both ends evenly rounded (Fig. 2, 17. 18, 33); anchored in single line, and possibly in pairs (Fig. lo), to oral bulge, some scattered in cytoplasm, never aggregated to warts as in some Loxophyllum species; form conspicuous layer in marginal fringe of cell, lacking only in anterior dorsal area

478 J. EUK. MICROBIOL.. VOL. 42. NO. 5, SEPTEMBER4K‘TOBER 1995

Table 1 . Morphonietric data of S/r.o/o.\ol,/n.//fc,,i itrrrcitlurrirc. populations.”

P0pUl;l- Character lion” I hl SD SD. CV Min Max n

Body. length

Body. maximum width

Macronuclear nodule. length

Macronuclear nodule. width

Micronucleus, largest diameter

Right lateral somatic kinetics. number (inel. right dorsolateral kine())

Left lateral somatic kineties. number (iiicl, dorsal brush row and left dorsolateral kinety)

Macronuclear nodules. number

Micronuclei. number

Contractile vacuoles. number

Berlin Munich Bonn Berlin Munich Bonn Berlin Munich Bonn Berlin Munich Berlin Munich Berlin Munich Bonn Berlin Munich Bonn Berlin Munich Bonn Berlin Munich Bonn Berlin Munich

133.0 135.0 23.8 3.6 146.2 140.0 20.8 3.7 199.6 9 28.9 8.3 31.0 29.5 7. I 1.1 54.6 55.0 10.8 2.0 53.8 5.4 1.7 10.7 11.0 I .5 0.2 13.9 14.0 I .5 0.3 14.8 3 2.5 0.7 7.8 7.5 1 . 1 0.2

10.8 10.5 -.- 3 1 0.4 2 . 1 -.- 1 7 0.5 0.1 2.9 3.0 1 .0 0.2

17.0 17.0 2.6 0.6 16.3 16.0 1.9 0.4 16.2 ? ? 6.7 7.0 0.7 0.1 6.2 6.0 1.2 0.2 6.0 6.0 0.0 0.0 7.0 2.0 0.0 0.0 7.1 2.0 0.5 0. I 7.0 2.0 0.0 0.0 I .0 1 .o 0.0 0.0 1 .o 1 .0 I .05 ? 2.0 2.0 0.0 0.0 2.0 2.0 0.0 0.0

’J

7

- - - -

17.9 65 I80 14.2 100 194 14.5 I65 264 22.9 16 45 19.8 30 75 10. I 44 62 14.0 8 13 10.9 I I 18 16.9 13 17 14.1 6 I I 20.0 7 16 19.4 I .5 4 35.4 2 6 22.0 I 1 14 11.9 I2 20

? 15 19 10.6 5 8 19.5 4 9 0.0 6 6 0.0 2 - 1

26. I 1 4 0.0 2 2 0.0 1 I

I 2 I 2

0.0 2 2 0.0 2 - 7

- -

44 29 12 44 29 12 40 31 14 40 31 32 29 18 30 I 1 36 29 1 1 41 30 20 41 30 20 22 20

Data based on randonil) selected. prorargol-impregnated and mounted specimens from exponentially growing cultures (Berlin and Munich populations) and field material (Bonn population). Measurements in pm. CV. coefficient of variation in %; M. median; Max, maximum; Min, minimum: n, number of observations: SD. standard deviation: SDx. standard deviation of the mean; X, arithmetic mean.

Data of Bonn population from 14 I ] ; very likely incorrect. at least partially, because the length of the figured, protargol impregnated specimens is smaller (142 pm) than the minimum value (165 pm) provided! Similar discrepancies exist with the number of somatic kineties.

where oral bulge is absent (Fig. 1. 16. 18. 19): stain heavily with silver carbonate (Fig. 16). but not with protargol.

Cortex thin. highly flexible. without special granules. e.g. mu- cocysts. Cytoplasm colourless. in central region of cell more or less densely filled, depending on food supply. with brightly shin- ing fat globules and food vacuoles; n o special cytoplasmic crys- tals. Feeds on small and medium si7ed ciliates (G1aitcor)ru sciti- tilluns, Cirrctochiliu)i rriurguri~uc~cuni, Colpidiirtti colpoda) and probably also on bacteria and/or detritus. Moves slowly, glides with densely ciliated right side on flat substrates or crawls ele- gantly on and between detritus aggregations showing great flex- ibility and deformation of bod)

General plan of somatic and oral infraciliature as in other members of order [ 131. In an old culture. most specimens had greatly reduced numbers of kineties. viz. 4-7 on right and 3 - 4 on left side. while body size was not markedly reduced.

Right side densely ciliated, kinetics with cilia about 7 p m long successively shortened along anterior half of perioral kinet) 3 and in posterior region of cell. where somc abut to right dor- solateral kinety and third perioral kinety. Right dorsolatcral kinety very near to dorsal margin of cell. bears regular somatic cilia and fibrillar associates. commences at anterior end of cell and curves around its posterior end (Fig. 6. 11, 11). Left side more sparsely ciliated than right. its ciliature consists of somatic kinctics. a brush kinety, and a dorsolateral kinety (Fig. 3. 7. 8. 12. 22. 28-30). Somatic kineties in central third of cell on top ofcortical crests. distinctly shortcned anteriorly and posteriorly. cilia reduced to 1-9 pni long stumps and about twice as widely spaced as on right side. Brush kinety in anterior third of body between leftmost somatic cilia? row and left dorsolateral ki-

nety. about 4 pm apart from dorsal margin of cell, consists of 30-50 very closely spaced dikinetids having 1-3 pm long cilia in anterior third of cell and ofclosely spaced, nonciliated mono- kinetids in posterior portion (“tail”), which extends left of a flat cortical crest (cp. Fig. 7.28); anterior portion ofbrush on bottom of depression formed by anterior end of oral bulge. often frag- mented, right fragments sometimes connected with crest kine- tics; cilia of dikinetids cylindroid to slightly inflated distally, anterior cilium usually slightly longer than posterior, length of cilia decreases from anterior to posterior (Fig. 28, 29); dikine- tidal axis usually parallel to main body axis, rarely oblique or almost transverse, especially if anterior portion is fragmented. Left dorsolateral kinety very near dorsal margin of cell. extends along its whole length and is thus continuous with perioral kinety 1 at both ends, bears about 2 p m long cilia and is thus easily distinguished from the almost adjacent right dorsolateral kinety which has regular (long) somatic cilia (Fig. 7 , 13, 30).

Oral bulge surrounding almost entire cell. leaving blank only small area a t anterior end of dorsal side (Fig. I , 3, 12, 13, 20, 29). about 2 pm high and thus difficult to recognize in the light microscope (Fig. 15, 18, 20). Anterior end of bulge curved to left surface of cell, producing inconspicuous crest right of which brush kinety commences. Bulge surface patterned string-like, with small hemispherical structures between riffles, possibly tips of toxicysts (Fig. 10). Nematodesmata very fine, originate from barren basal bodies of perioral dikinetids (see TEM section), recognizable only up to mid-body, indicating that functional mouth is much shorter than oral bulge. Perioral kinety 1 a t left margin of oral bulge, merges into left dorsolateral kinety ante- riorly and posteriorly, anterior half composed of regularly spaced

FOISSNER & LEIPE-SSIROLO.~OPIIYLLU~~ N. G 479

dikinetids, posterior portion made of equidistantly spaced monokinetids; anterior basal body of dikinetids lighter stained than posterior one, bears about 2 pm long, cylindroid ciliary stump (Fig. 3, 8, 12, 13, 27, 29, 30); dikinetids orientated obliquely to kinety axis, i.e. parallel to main body axis, without kinetodesmal fibres in silver carbonate impregnated specimens (Fig. 26). Perioral kinety 2 at right margin of oral bulge, com- posed of tightly spaced dikinetids, at least in anterior half, as indicated by distribution of nematodesmata; anterior or pos- terior basal body of dikinetids with regular, about 7 pm long cilium; dikinetids orientated obliquely or almost transversely to main body axis, without kinetodesmal fibres in silver car- bonate stains (Fig. 6, 11, 17, 19, 26). Perioral kinety 3 right of and very close to kinety 2, ends indistinctly separate from right dorsolateral kinety, composed of monokinetids throughout; ki- netids with normal long cilia and conspicuous kinetodesmal fibres orientated more laterally than those of somatic kinetids. at least in anterior half (Fig. 6, 1 1, 17, 19, 24).

Transmission electron microscopy. The fine structural in- vestigations are not very detailed because they were not the main objective of the study. Thus, the description will be brief, emphasizing some new findings.

The somatic kinetids of S. utriculuriue have typical haptond pattern, including two transverse microtubular ribbons (Fig. 3 1). The first transverse ribbon extends obliquely anteriad and is longer than the second ribbon, which extends transversely (ra- dially). Both ribbons originate near triplets 3-5. The postciliary microtubules are very long and form distinct stripes recogniz- able in protargol stains (Fig. 9, 3 1).

The string-like pattern of the oral bulge is conspicuous also in ultrathin sections (Fig. 32). The riffles contain the transverse microtubular ribbons originating from the nonciliated basal bodies of the perioral dikinetids (Fig. 32-35). There is no per- manent oral opening.

Perioral kinety 1 is composed of oblique dikinetids. The an- terior basal body bears a short cilium and inconspicuous post- ciliary and transverse microtubular ribbons; the posterior basal body is not ciliated and associated with a conspicuous nema- todesma and a transverse microtubule lamella extending into the oral bulge (Fig. 7, 33-35). Perioral kinety 2 consists of di- kinetids as kinety I . Its structure could not be unequivocally clarified. One basal body of the dikinetids, possibly the anterior. is nonciliated and associated with a nematodesma and a long transverse microtubule ribbon extending into the oral bulge; the other basal body bears a normal long cilium (Fig. 25) and is possibly associated with a postciliary and/or transverse micro- tubule ribbon (Fig. 32-35). The kinetids of perioral kinety 3 are ciliated and look like somatic kinetids, except of the kineto- desmal fibres which extend more obliquely (Fig. 6, 17, 24, 25, 32). They form typical triads with the dikinetids of perioral kinety 2 (Fig. 32), as described by Bohatier & Njine [2] in Li- tonotus.

Divisional morphogenesis. Stomatogenesis and cell division of S. utriczilariae proceed as described by Fryd-Versavel et al. [24] in Arriphilqtus pleut-osigma. We thus provide only a sum- mary of our observations. Proliferation of basal bodies occurs intrakinetally in all kineties, migrating kinetofragments do not occur, and the parental infraciliature is apparently retained un- changed, The most conspicuous event is the appearance of paired brush cilia close underneath the prospective division furrow (Fig. 23). These cilia very likely grow out from the nonciliated monokinetids found in the rear (“tail”) of the parental brush kinety (Fig. 7). The new tails produced in the proter and opisthe are also barren. Thus, the brush kinety shows a nonciliated pre- equatorial and posterior portion throughout the entire division process (Fig. 23). How the dikinetids for the opisthe are pro-

Fig. 1-5. Siroloxophylliitrr utriculuriue from life (Munich popula- tion). 1. Right lateral view of typical specimen. Scale bar division = 20 pm. 2. Extrusomes are 6-8 pm long and curved. 3. Anterior end of left side. The oral bulge (arrow) ends close to the top of the cell, leaving blank only a small area at the anterior dorsal margin. 4, 5. Dorsal and transverse view showing flattening of cell. The left surface is distinctly furrowed and bears shortened cilia. B, dorsal brush; CV, contractile vacuoles; E, extrusomes; FV, food vacuole; LK, left lateral somatic kineties; MA, macronuclear nodule: OB, oral bulge; PI, perioral kine- ty 1.

duced, i.e. by rearrangement oftail monokinetids or by addition of new basal bodies to existing monokinetids. could not be ascertained and needs TEM investigation of dividing specimens.

The macronuclear nodules fuse and the micronucleus divides during the early stages of stomatogenesis, i.e. before the division furrow is recognizable. After the division furrow has appeared, the roundish macronuclear mass divides into two nodules which migrate into the proter and opisthe, respectively, where they divide again to produce the interphase pattern.

Ecology. This section is a compilation of the faunistic and ecological literature available on S. utriculuriae. Few records are known, most are from running and stagnant freshwaters; those from mosses and soils in Germany [48] and New Zealand [43, 441 are very likely misidentifications, because the species died in our cultures without forming permanent (resting) cysts, indicating that it cannot live in soil. Furthermore, we have never found it in the more than 1,000 soil and moss samples inves-

FOISSNER & LEIPE-SIROLOXOPHYLLUM N. G. 48 1

Fig. 14-20. Siroloxophyllum utriculariae. 14. from life. 15, 18, 20. fixed as for scanning electron microscopy. 16, 17, 19. silver carbonate impregnation (14, 16, 17, 19 from Berlin population; 15, 18, 20 from Munich population). 14, 15,16, 18. Right and left lateral views. Extrusomes are concentrated in hyaline fringe surrounding cell. Arrows mark contractile vacuoles, arrowheads indicate macronuclear nodules. 17, 19. Oral and somatic infraciliature. Kinetodesmal fibres of kinetids of perioral lunety 3 are more laterally directed than those of somatic kinetids. Arrowhead marks right dorsolateral kinety. 20. The anterior end of the oral bulge (arrow) is curved to the right at the left side of the cell. Arrowheads mark small region between ends of oral bulge (cp. Fig. 29). B, dorsal brush; CV, contractile vacuole; E, extrusomes; F, fringe; KD, kinetodesmal fibres; MA, macronuclear nodules; PI , 2, 3, perioral kineties; RK, right lateral somatic kineties. Bars in 14-16 = 60 pm. Bars in 17-20 = 20 pm.

482 J. EUK. MICROBIOL.. VOL. 42, NO. 5 . SEPTEMBERaCTOBER 1995

Fig. 21-25. S / r o / o . u o ~ ~ ~ l , / / u r f i ctlnczr/arrue. SEM micrographs (2 1-24. Berlin population; 25, Munich population). 21, 22. Right and left lateral view of morphostatic cells. 22 shows a well-fed specimen lacking furrows on left side (cp. 23. 28). Arrowheads mark excretory pores of contractile vacuoles (cp. 24); arrows indicate left lateral somatic kineties having very short cilia. 23. Middle divider. Arrows mark posterior (proter) and anterior (opisthc) end of dorsal brush. respectively. 24. Each contractile vacuole has many excretory pores (arrows). 25. Right anterior end showing that pcrioral kineties 2 and 3 arc ciliated. B. dorsal brush: PI, 2. 3. perioral kineties. Bars in 21-23 = 50 prn. Bars in 24, 25 = 10 pm.

Fig. 26-30. Siroloxophyllum utriculuriae. 26. silver carbonate impregnation. 27-30. SEM micrographs. Inset in 30 is light micrograph of fixed specimen. (26-28 from Berlin population; 29-30 from Munich population). 26,27. Oral infraciliature. Perioral kineties I and 2 consist ofdikinetids having only anterior or posterior basal bodies ciliated (cp. 6, 7). 28, 29. Left anterior ends of strongly furrowed specimens. The length of the dorsal brush cilia decreases gradually from anterior to posterior and no ciliary stumps are recognizable in the furrow extending posteriorly of the brush kinety although basal bodies are present (cp. Fig. 7). The oral bulge surrounds almost the entire cell, leaving only a small area at the anterior dorsal side blank (arrows). 30. Left posterior end in the light (inset) and scanning electron microscope. Perioral kinety I and left dorsolateral kinety are continuous and the oral bulge has a distinct string-like pattern recognizable even in the light microscope (arrows). B, dorsal brush; LD, left dorsolateral kinety; LK, left lateral somatic kineties; OB, oral bulge; PI, 2, 3 , perioral kineties. Bars in 26-29 = 10 pm. Bar in 30 = 5 prn.

484 J . EUK. MICROBIOL., VOL. 42. NO. 5 . SEPTEMBER4CTOBER 1995

Fig. 31-35. S ~ r o / o , ~ o p h ~ ~ / / u ~ ~ z utriculur/ae. TEM micrographs from Berlin population. 31. Tangential section of right side showing ultrastructure of somatic conex and monokinetids. 32. Oblique longitudinal section showing riffles of oral bulge and triads formed by kinetids of penoral kineties 2 and 3.33-35. Oblique serial section of oral area showing details of kinetids from perioral kineties 1 and 2. BB, basal body; E, extrusome (toxicyst): KD, kinetodesmal fibre; N. nematodesma; PC. postciliary microtubular ribbon; PI, 2. 3. perioral kineties; R, riffles of oral bulge; RK, right lateral somatic kinety: T. transverse microtubular ribbons of oral kineties; TI. T2, transverse microtubule ribbons of somatic monokinetids. Bars = 1 um.

FOISSNER & LEIPE-SIROLOXOPHYLLUM N. G. 48 5

tigated during the last decade. Thus, all reliable records are from freshwaters of central and eastern Europe and Mexico [40]. It seems that S. utriculariae is a rare species, usually occumng with low abundance.

Penard [36] and Kahl [27, 281 found S. utriculariae between Utricularia weed in Geneva (Switzerland) and Hamburg (Ger- many), respectively. Several records [7, 111 are available from the Danube river, where S. utriculariae lives in the periphyton of stones, and from oligosaprobic and mesosaprobic rivers, brooks and ponds in Germany [3,22], Bulgaria [6,7] and Mexico [40]. Detcheva [6] provides the following abiotic parameters from a single record in a beta-mesosaprobic river in Bulgaria: pH 7.7,8 mg/L O2 (94% saturation), 3.6 mg/L biological oxygen demand (5 days), 12.6 mg/L chemical oxygen demand, 118 mg/LCa2+, 24 mg/LMg2+, 0.25 mg/LNH,+-N, 1.9 mg/LNO,-- N, 0.06 mg/L NO,--N, 0.2 mg/L Fez+, 0.2 mg/L Mn2+, 0.06 mg/L phenols. We found S. utriculariae infrequently and with low individual numbers in beta-mesosaprobic to alpha-meso- saprobic rivers near Munich, Germany ([22] and Fig. 36). It occurred more regularly and abundantly in the sludge of rapid gravity filters of some waterworks in this region; the abundance variations observed could be not correlated with specific biotic and process parameters [ 181.

Siroloxophyllum utriculariae glides slowly and elegantly in the periphyton of natural and artificial substrates. Like other members of the group it is a predator. However, detailed ob- servations from natural populations are not available. In cul- tures it feeds on small to medium-sized ciliates, like Glaucoma scintillans and Colpidium colpoda, which are apparently quickly digested because the cells are usually rather hyaline and rarely contain identifiable prey residues; bacteria and/or organic de- tritus are probably also ingested. Biomass of lo6 medium-sized (150 x 50 x 20 pm) cells about 90 mg [23]. SladeEek et al. [39] and Wegl [47] consider S. utriculariae as an excellent indicator of beta-mesosaprobic conditions and provide the following va- lency spectrum: beta-mesosaprobic; oligosaprobity (0) = 1, be- tamesosaprobity (b) = 8, alpha-mesosaprobity (a) = 1, indica- tion weight (I) = 4, saprobity index (SI) = 2.0. However, the data available indicate that the oligosaprobic proportion should be increased in the valency; but this needs further investigations ~ 3 1 .

DISCUSSION Siroloxophyllurn as a new genus. Kahl [28] transferred Am-

phileptus utriculariae [36] to Loxophyllum. This was accepted by Song & Wilbert [41], who reinvestigated the species using protargol impregnation (Fig. 43-48), Our investigations show that A. urriculariae belongs neither to Amphileptus nor Litonotus (because it lacks a median suture and has a right dorsolateral kinety) nor to Loxophyllum, whose left anterior end is occupied by a conspicuous field of paired brush cilia [23] which was overlooked by Song & Wilbert [41].

The most conspicuous character of Siroloxophyllum is the string-like patterned oral bulge surrounding almost the entire cell, leaving blank only a small area at the anterior dorsal end (Fig. 3. 12, 29). This feature is not easily recognized in living and protargol impregnated cells. However, if one is aware of its existence, it can be seen well under interference contrast (Fig. 20). Recent SEM observations showed that the oral bulge of very likely all pleurostomatid ciliates is patterned string-like [23]. The distinctiveness of the pattern varies; usually it is most conspicuous in suboptimally prepared specimens. Thus, the pat- terned oral bulge of Siroloxophyllum is not unique, but it is exceptional in surrounding almost the entire cell. It is not known whether S. utriculariae can open the whole bulge during feeding

4,5 51 - .- E 3.5 4l TO”,

Fig. 36. Frequency and rated abundance [semi-logarithmic scale: I (rare), 2, 3, 5, 7, 9 (very numerous)] of S. urriculuriue in 379 samples collected during 1987-1 991 in beta- to alphamesosaprobic Bavarian streams.

or-like other members of the family 1231-only that portion which is accompanied by the paired basal bodies of perioral kineties 1 and 2. Likewise, the mechanism which unlocks the bulge between perioral kineties 1 and 2 is obscure. Possibly, the transverse microtubular ribbons of the oral kinetids are in- volved.

An even more difficult character is the dorsolateral kineties. A left dorsolateral kinety is very likely present in all pleuro- stomatids (although often not designated or recognized as such), possibly with the exception of Loxophyllum meleagris, and lo- cated between the dorsal brush kinety and the rightmost somatic ciliary row or the right dorsolateral kinety [19, 231. The left dorsolateral kinety, which was considered as regular left lateral somatic ciliary row by most previous authors, differs clearly from the left lateral kineties by being continuous with the mono- kinetidal tail of perioral kinety 1 (Fig. 7,30); from the rightmost somatic ciliary rows of the right side and from the right dor- solateral kinety it differs by the short, stump-like cilia (Fig. 28, 30). Right dorsolateral kineties are present only in Loxophyllum, which has two [23], and in Siroloxophyllum. which possesses only one (Fig. 6, 19). The right dorsolateral kinety(ies) differs from the right lateral ciliary rows by surrounding the posterior end of the cell, forming a more or less distinct suture with the abutting ends of the regular somatic kineties.

The structure and/or location of the dorsal brush of Siroloxo- phyllum differ distinctly from Loxophyllum, Pseudoamphileptus and Opisthodon, but are similar to Litonotus, Acineria and Am- phileptus (Fig. 49).

Thus, none of the four characters given in the genus diagnosis is unique to Siroloxophyllurn, i.e. it is only the specific com- bination of the characters which separates the new genus from its relatives.

Species assignable to Siroloxophyllum. A reinvestigation of the protargol impregnated type slides of Loxophyiium australe [ 191 showed that it has the main characteristics of S. utriculariae. Thus, it has to be transferred to this genus: Siroioxoph.vlium austraie (Foissner & O’Donoghue, 1990) nov. comb. The two species differ mainly in the number of macronuclear nodules, usually two in S. utriculariaeand four in S. australe. The number of right end left lateral somatic kineties is slightly higher in S. utriculariae than in S. australe. Very likely, other species will be added, e.g. Loxophyllum carinatum Vuxanovici and L. semi- h a r e Vuxanovici (both redescribed in [4 11, but seemingly with- out dorsolateral kineties and thus not definitely assignable).

486 J. EUK. MICROBIOL.. VOL. 42. NO. 5. SEPTEMBER-OCTOBER 1995

Fig. 3 7 4 8 . Published dramings of S. ~rrricxluriilc. 37-39. Left lateral and dorsal view and nuclear apparatus from life, length 65-120 pm. Arrows mark contractile \acuoles. From (361. 40. Leli lateral view from life. length 100 jm. From (271. 41. Left lateral view from life, length I50 pm, Arrows mark contractile \acuoles. From [XI . 42. Left lateral view from life. siie not indicated. Arrows mark contractile vacuoles. From [40]. 4 3 4 8 . Left (43. 46). right (45) and ventral (48) views from life (43) and after protargol impregnation (45. 46. 48); extrusomes (44) from life, silverline sqstcm (47) after dry sil\cr nitrate impregnation. From (4 I]. B. dorsal brush: CV. contractile vacuoles; MA. macronucleus: MI, micronucleus: 0s. oral slit (mouth entrance): PI . 2. 3. perioral kineties.

Comparison of descriptions of S. utriculuriue. Our obser- vations basically agree with those mentioned in the original description [36] and the two redescriptions [28, 411. Thus. we d o not doubt the identification and conspecifit! of all popula- tions. However. some differences should be noted. Penard [36] drew the anterior contractile vacuole near the dorsal margin and the posterior kacuole near the ventral side (Fig. I , 21). whereas Kahl [28] and Song & Wilbert [41] definitely stated an opposite location (Fig. 41. 43. 45). which agrees with our observations (Fig. I . 14. 18. 21). Thus. i t may be assumed that Penard’s indication is a simple mistake. all the more so as he did not definitely describe the location of the vacuoles. Another differ- ence concerns the extrusomes which. according to Penard [36]. are elongated in the strongly flattened and slightly protruding oral area (Fig. 37). whereas Kahl [78] and Song & Wilbert [41] found them to be of the same length over the whole perimeter of the cell (Fig. 41. 43). which matches our observations (Fig. I . 16). Although the shape and s i x of the extrusonies are im- portant species characteristics in gymnostomatid ciliates [ 13. 231, this difference cannot be weightened heavily because Penard [36] never used oil immersion objectives and thus v e n likely could not ascertain the real length of the extrusomes in the thicker, opaque parts of the cell.

Song & Wilbert [4 I] redescribed S . irfric~irluuue very briefly. but provided some elegant drawings (Fig. 43-48) which, how- e\er . d o not give an! indication of dorsolateral kineties. We suppose that Song & Wilbert overlooked them because their description contains a k o other unfortunate mistakes. They fig- UI‘C a l l oral kineties as being composed of dikinetids and the oral slit between perioral kineties 7 and 3 (Fig. 48). Both ob-

servations are clearly disproved by our data (Fig. 8. I I , 32-35) and literature evidence [ 2 , 13, 231.

Ultrastructure. The fine structure of the somatic kinetids of S. ri/ricir/ariacis very similar. ifnot identical. to that ofhaptorids like S p l h i d i r t ~ [49] and Eirc.h~/vdizirn [20]. The second trans- verse microtubular ribbon was apparently overlooked in pre- vious descriptions of plcurostomatids, but can be recognized in published micrographs of Lo.~oph~dhrr? rrieleaagris (Fig. 16, 17 in [37]). Sir.o/(j.\-c,ph!,//irrii irfririrlariae is thus a ditransversal ciliate in the sense of Leipe 6i Hausmann [32].

The interpretation of the oral structures is more difficult. As concerns perioral kinety I , our results agree with previous de- scriptions [2. 371. while the structure of perioral kineties 2 and 3 appears different in several respects. Whether these differences are genus specific or caused by interpretation problems needs further investigations. At least some data in Bohatier & Njine’s [ 2 ] paper appear doubtful. for instance that perioral kineties 2 and 3 lack cilia. In S. iitricdar-iue they are ciliated (Fig. 25) and form the typical mane recognized earlier by Kahl [28] in many pleurostomatids. A second problem is posed by the kinetodes- ma1 fibre. which is. according to Hohatier & Njine [2]. associated with the posterior basal body of the dikinetids of perioral kinety 2. Our data show that it originates from the monokinetids of perioral kinety 3 (Fig. 17. 19). which is more likely since the haptorid oral dikinetids generally lack a kinetodesma [34]. Peri- oral kinety 3 is very likely a specialized somatic kinety, anal- ogous (because it apparently lacks nematodesmata) to the or- alized somatic kinetids found in several haptorids [2 I ] . A third problem concerns the species investigated by Bohatier & Njine [?I. Their figures doubtlessly show a Lifotiofits species, as in-

FOISSNER & LEIPE-SIROLOXOPHYLLUM N . G 487

) 3 , 2

!:I .* .* .. .. .* .. .*

L i t o n o t u s

f

? ? I I I-- 6 I I

P2

0

I

Acinexia

R

Sirol o x o p h y l 1 urn Loxoph y l l um

Amph il e p t u s P s e u d o a m p h i l e p t u s O p i s t h o d o n

Fig. 49. Genus distinction in the order Amphileptida by the arrangement of the right lateral ciliary rows (with spica in Amyhileptus, Pseu- doamphileptus. Opisfhodon), the number of perioral kineties (three in Litonotus, Acineria, Siroloxophyllum, Loxophyllum; two in others), the presence (Siro/ox-ophy//um, Loxophyllurn)/absence of right dorsolateral kineties, the shape of the anterior body end (curved in .4cineria, arrow), the dorsal brush (large field in Loxophyllum, pocketed in Opisfhodon, very near oral bulge in Pseudoamphileptus, single row in others) and the length of the oral bulge (extending to posterior end in Loxophyllum and Pseudoamphileptus, surrounding cell in Siroloxophyllum, extending to mid-body in others). Note that Pseudoamphileptusand Opisfhodon are still insufficiently defined, i.e. need redescription based on better impregnated specimens. Heininofus is excluded because its infraciliature is not known. B, dorsal brush; OB, oral bulge: P1, 2 , 3, perioral kineties; RD, right dorsolateral kinety.

488 J . EUK. MICROBIOL., VOL. 42, NO. 5 . SEPTEMBER-OCTOBER 1995

dicated by the triads formed by the kinetids of penoral kineties 2 and 3, but very likely not L. quadrinuclearus which lacks. according to Dragesco & Njine [8], penoral kinety 3 but has a conspicuous spica, indicating that it belongs to the genus . 4 m - phileptus [ 131. Unfortunately. Bohatier & Njine [2] did not men- tion the source of their material.

Systematic relationships of Siroloxophyllum and classifica- tion of pleurostomatid ciliates. Traditionally, all pleurosto- matid genera are lumped in a single family, Amphileptidae Butschli [4, 5. 281. However, Foissner & Foissner [ 2 I] split the pleurostomes into two suborders, viL. Amphileptina and Liton- otina and recognized two families. viz. Amphileptidae and Li- tonotidae. More recently. Lipscomb & Riordan [34] suggested a very different classification based on cladistic methods, using. however, many unproven character states. They assigned to the pleurostomes not only lacrymariids and didiniids but also clas- sical haptorids like Spathidium, Bvophyllum and Homalozoon. We believe that this was an unsuccessful upset, simply because the distinct asymmetry of the pleurostomatid oral and somatic ciliature is hardly found in any classical haptorid, with the no- table exception of Homalozoon, a highly thigmotactic and spe- cialized predator. Furthermore, Lipscomb & Riordan [34] did not take into account the different types of stomatogenesis oc- cumng in pleurostornes s. str. (monotelokinetal) and haptorids s. str. (holotelokinetal; see [33] for definition of terms and lit- erature). Obviously, their classification neglects two main fea- tures and is thus very likely artificial.

The classification suggested here thus follows Foissner & Fois- sner [21] and includes the data mentioned in their publication and in the present study.

Order Pleurostomatida Schewiakoff, 1896 Oral area flattened along ventral margin of laterally com-

pressed body, surrounded by toxicysts; rhabdos made of three microtubular components: transverse ribbons originating from the oral dikinetids and in suborder Litonotina also from somatic monokinetids, nematodesmal bundles originating exclusively from oral dikinetids. and bulge microtubules; somatic ciliature with distinct left-right differentiation, including dorsal brush and, in some genera, one or two dorsolateral kineties; free-living and parasitic on other ciliates (mainly peritrichs), often large. lengthy voracious carnivores; widely distributed in freshwater. marine, and interstitial habitats. Type: Amphileptina Jankows- ki, 1967 [26].

Suborder Amphileptina Jankowski. 1967 Cytostome surrounded by a right and a left perioral kinety

composed of dikinetids; right somatic ciliature with spica. Type: Amphileptidae Butschli. 1889 [4].

Remarks: This suborder is monotypic. i.e. includes only the family Amphileptidae Biitschli with the characteristics given for the suborder. The genera Amphileptus [lo. 13; type by virtual tautonymy], Opisthodon [ 13, 421 and Pseudoamphileptus [ 121 belong to this family. Hcmiophrys [50] is a junior synonym of Amphileptus [ 131. .4mphileptus carchesii Stein very likely needs a separate genus, because it has a heavily ciliated groove which secretes a loop-like structure anchoring the ciliate to the prey [23]. However, the separation should await a detailed study of its in fraci lia turc.

Suborder Litonotina Foissner & Foissner. 1988 Cytostome surrounded by a right and left perioral kinety com-

posed of dikinetids, right kinety accompanied by (oralized ?) somatic rnonokinetids forming a distinct 3rd perioral kinety

dos; right lateral ciliature with or without dorsolateral kineties. ciliary rows successively shortened along perioral and dorso- lateral kineties. Type: Litonotidae Kent, 1882 [3 I].

Remarks: This suborder includes the families Litonotidae Kent and Loxophyllidae n. fam., differing mainly by the absence/ presence of right lateral dorsolateral kineties.

Family Litonotidae Kent, 1882 [3 11: Litonotina without right dorsolateral kineties. Type: Litonotus Wrzehiowski, 1870 [50].

Remarks: This family includes the genera Litonotus [ 13, 501, Acineria [ 1. 91 and, possibly, Heminotus [29] whose infracilia- ture has been not yet described.

Family Loxophyllidae n. fam.: Litonotina with dorsolateral kineties. Type: Loxophyllum Dujardin, 184 1 [9].

Remarks: Jankowski also mentioned a new family “Loxo- phyllidae” without, however, providing any characterization or type genus (Jankowski, A. W. 1975. A conspectus of the new system of subphylum Ciliophora Doflein, 190 1. Abstract. In: Balashov, U. S. (ed.), Account of Scientific Sessions on Results ofscientific Work, Year 1974: Abstracts ofReports. Akad. Nauk SSSR, 2 0 1 . Inst. Leningrad. Pp. 26-27 [in Russian]). Thus, the name is illegitimate, i.e. not in accordance with the rules of nomenclature. According to Grain [25], Dujardin [9] also found- ed a family Loxophyllidae. However, this is not confirmed by an inspection of the original literature. Dujardin [9] included Lo.rophyllum and other pleurostomes in his new family Para- meciidae.

The family includes two genera, viz. Loxophyllum [9, 231 and Sirolo.uophyNum n. g . Lo-rophyllum was formerly [2 11 assigned to the Amphileptina because the data available suggested that it lacks perioral kinety 3. This was disproved by a reinvestigation [23]. Very likely, some ofthe many marine and interstitial Loxo- phyllum species are not congeneric. Unfortunately, their infra- ciliature is not known and any separation would be premature.

Key to pleurostomatid genera. The following key uses data from the present study and the literature [ 1 , 12. 13, 19, 23, 281. No reliable information is known from Heminotus, which is thus excluded. For definition of specific morphological terms see Foissner [ 131, material and method section and Fig. 49. Differences between some genera are clearly recognizable only after protargol impregnation. Likewise, the proper generic clas- sification of most species requires protargol impregnation or at least careful examination of living specimens with interference contrast.

1. Two perioral kineties. Right side somatic kineties shortened in midline of cell, forming more or less distinct suture (spica) in

Three perioral kineties. Right side somatic kineties shortened anteriorly and abutting to perioral kinety 3. With or without

2. Left anterior end smooth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Left anterior end with small cavity containing anterior end of

dorsal brush kinety . . . . . . . . . . . . . . . . . . . . . . . . . . . . Opisthodon 3 . Oral bulge indistinct. Dikinetidal portion of perioral kineties

extends to mid-body . . . . . . . . . . . . . . . . . . . . . . . . . Amphileptus Oral bulge distinct. Dikinetidal portion of perioral kineties ex-

tends near posterior end of cell . . . . . . . . . . . Pseudoamphileptus 4. With right dorsolateral kineties. Single brush kinety or dcnsc

5 Without right dorsolatcral kineties. Single brush kinety . . . . . . . 6

5. Oral bulge extends along ventral side. Two right dorsolateral kineties. Many brush kineties continuous with anterior end of left lateral kineties . . . . . . . . . . . . . . . . . . . . . . . . . . Loxophyllurn

Oral bulge surrounds almost entire cell. Single dorsolateral ki- nety. Single brush kinety at dorsolateral margin of cell

anterior half of ciliate. Right dorsolateral kineties absent . . . 2

right dorsolatcral kineties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

field of short, paired cilia in anterior region of cell . . . . . . . . .

. . . . whose transverse microtubular ribbons contribute to the rhab- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Siroloxophldlum

FOISSNER & LEIPE-SIROLOXOPHYLLUM N. C. 489

6. Oral bulge and perioral kineties terminate at anterior end ofcell

Oral bulge and perioral kineties distinctly curved to left anterior Litonotus

surface of cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acineria

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ACKNOWLEDGMENTS Supported by the Bayrisches LAWW and the Austrian FWF

(Project PO 8924-BIO). The technical assistance of Dr. Andreas Untenveger, Mag. Margit Palzenberger, Andreas Zankl and Mag. Eric Strobl is greatly acknowledged.

LITERATURE CITED 1 . Augustin, H., Foissner, W. & Adam, H. 1987. Revision of the

genera Acineria, Trimyetna and Trochiliopsis (Protozoa, Ciliophora). Bull. Br. Mus. Nut. Hist. (Zool.). 52: 197-224.

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3. Buck, H. 196 I . Zur Verbreitungder Ciliaten in den Flieogewassern Nordwiirttembergs. Jh. Ver. vaterl. Naturk. U'urtt., 116: 195-2 17.

4. Biitschli, 0. 1887-1 889. Protozoa. Abt. 111. Infusoria und System der Radiolaria. In: Bronn, H. G. (ed.), Klassen und Ordnung des Thier- Reichs. Winter, C. F.. Leipzig. 1: 1098-2035.

5. Corliss. J. 0. 1979. The Ciliated Protozoa. Characterization, Classification, and Guide to the Literature, 2nd ed. Pergamon Press, Oxford.

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7. Detcheva, R. B. 1992. Protozoa, Ciliophora. Cat. Faunae Bulg.,

8. Dragesco, J. & Njine, T. 197 1. Complements a la connaissance des cilits libres du Cameroun. Annls. Fac. Sci. Univ. j6d. Cameroun.

9. Dujardin, F. 184 1. Histoire naturelle des zoophytes. Infusoires. Suites a Buffon. Paris.

10. Ehrenberg, C. G. 1830. Beitrage zur Kenntniss der Organisation der Infusorien und ihrer geographischen Verbreitung, besonders in Si- birien. .4bh. dt. Akad. R'iss. B e d , year 1830: 1-88.

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13. Foissner, W. 1984. Taxonomie und Okologie einiger Ciliaten (Protozoa. Ciliophora) des Saprobiensystems. 1: Genera Litonotus, Am- phileptus, O~isthodon. Hydrohiologia. 119: 1 93-208.

14. Foissner, W. 1992a. Observing living ciliates. In: Lee, J. J. & Soldo, A. T. (ed.), Protocols in Protozoology. Society of Protozoologists, Allen Press, Lawrence, Kansas. Pp. C- 10. I-C- 10.2.

15. Foissner, W. 1992b. The silver carbonate methods. In: Lee, J. J. & Soldo, A. T. (ed.), Protocols in Protozoology. Society of Protozo- ologists, Allen Press, Lawrence, Kansas. Pp. C-7.142-7.4.

16. Foissner, W. 1992~ . Protargol methods. In: Lee, J. J. & Soldo, A. T. (ed.), Protocols in Protozoology. Society of Protozoologists, Allen Press. Lawrence, Kansas. Pp. C-6.1-C-6.8.

17. Foissner, W. 1992d. Preparation of samples for scanning elec- tron microscopy. In: Lee, J. J. & Soldo, A. T. (ed.), Protocols in Pro- tozoology. Society of Protozoologists, Allen Press, Lawrence, Kansas.

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7-8 :9 7- 1 40.

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Received 11-23-94; accepted 3-29-95

J Euk Microbrol. 42(5), 1995, pp 49C-505 0 1995 by the Society of P ~ O I O Z W ~ O ~ S I S

Ultrastructure of the Parabasalid Protist Holomastigotoides WILMA I,. LINGLE' and JEFFREY L. SALISBURY

Depur/mertt qf Biochein/s/rv and .Cfoleculur Bioloy?,, .Way0 Clinic and Foundation. Rochessrer. Minnesota 5590s

ABSTRACT. The ultrastructure of two specics of Ilolomostrgotoides is presented. The basic unit of organization of these large cells is the flagellar band. Each flagellar band consists of a row of flagellar basal bodies linked by three fiber systems. The number of flagellar bands is species dependent. The flagellar bands originate at the cell apex and are arranged in parallel spirals of increasing gyre, thus defining the conical shape of the cell. In the cell apex a striated root called a parabasal fiber is juxtaposed with the basal bodies of each flagellar band. Linear extensions of two parabasal fibers function as the spindle poles for the persistent extra-nuclear spindle. The nucleus is in close contact with the spindle poles and spindle microtubules. Parallel sheets of microtubules which constitute axostyles are nucleated along the underside of the parabasal fibers. The axostyles extend away from the cell apex, with many reaching the basal region of the cell. Some of the axostyles follow the spiral pattern of the flagellar bands. Numerous Golgi bodies are spaced regularly along the flagellar bands. Together the parabasal fiber. axostyles and Golgi bodies associated with a flagellar band are termed a parabasal complex.

Supplementary key words. Basal bodies. cytoskeleton. fibers. mitotic spindle, parabasal complex, parabasalid, spindle poles, TEM.

OLOM.4STIGOTOIDES spp. are parabasalid protista H found in the hindgut of four genera of primitive termites, Prorhinotertnes, Coptotertncs, Hctcrotertncs. and Psamtno- termes, where they aid in digestion of wood [2. 3, 14, 18, 231. Like all parabasalids, they have parabasal complexes, each of which is comprised of a Golgi complex and a striated root called a parabasal fiber, associated with flagellar basal bodies. The parabasal complex is the primary distinguishing characteristic of members of the Class (or Phylum, depending on classification scheme) Parabasalia of zooflagellate protists [ 1 , 9, 10, 161. Re- gardless of the hierarchical level of the taxon, the Parabasalia are considered a monophyletic group based on the conserved presence of parabasal complex, hydrogenosomes (organelles in which anaerobic metabolism occurs [ 191). and axostyles, and the absence of mitochondria. There are two parabasalid orders: the Trichomonadida and Hypermastigida. Hypermastigotes tend to be larger and have more extensive flagellated regions than trichomonads. The flagellated regions consist of rows of flagella often arranged along bands that cover most of the cell surface or in large apical clusters [ 1, 91.

In the hypermastigote H. tusitala the extended flagellated regions consist of five bands of flagella which originate a t the anterior apex of the cell and spiral posteriorly through approx- imately 5.5 gyres of increasing diameter, thus defining the con- ical shape of each cell [4, 51. T h e smaller H . diversa, also found in P. simplex, has 8 flagellar bands, but fewer gyres. Previous light microscopy descriptions of H. tusitala and H . diversa, the focus of our work presented here, demonstrated the presence of two to four large chromosomes which are condensed during most of the cell cycle. a n extra-nuclear, persistent mitotic spin- dle, linear spindle poles attached to two flagellar bands. and kinetochores a t the nuclear envelope [4-81. Limited transmis- sion electron microscopic observations have been made on sev- eral other species of Holomastigotoides [ 1 1, 15, 221. Gibbons

' To whom correspondcnce should be addressed.

and Grimstone [ 1 I] described and compared the ultrastructure of flagellar basal bodies of Holornastigotoides sp. with those of two other hypermastigote genera, Pseudotrichonytpha and Tri- chonytnpha. The two latter genera have longitudinal rows of flagella (12.000-1 4,000 flagella in T . carnpanula) rather than the spiral arrangement of flagellar rows of H. spp. The hyper- mastigotes, including H. hcrnigytnnurn, were the subject of a comparative structural analysis of the mitotic apparatus and cell division [IS]. Our a im in the present work is to provide a com- prehensive ultrastructural description of two species of Holo- rnastigotoides found in the termite P. sitnplex, with special re- gard to cell polarity and transitions in the cytoskeleton and parabasal complex.

MATERIALS AND METHODS The hindgut was removed from P. sirnplex workers and the

contents were placed immediately in fixative consisting of 5% glutaraldehyde (Electron Microscopy Sciences, Fort Washing- ton, PA) in 0.025 M HEPES (Sigma Chemical Co., St. Louis, MO), p H 7.0 for 2 h on ice. After washing three times for 5 min, the fixed cell suspension was pelleted and embedded in a drop of 4% agarose (Sigma). The hardened agarose pellet was diced into small pieces prior to further handling. The cells were post-fixed for 1 h o n ice in I % osmium tctroxide (Electron Microscopy Sciences, Fort Washington, PA), O . ~ O / O potassium ferrous cyanide (Fisher Scientific Co., Fair Lawn, NJ) in 0.2 M potassium phosphate buffer, pH 7.0. After washing with deion- ized water for 5 min, the cells were treated with 0.15% tannic acid (Fisher Scientific Co.) in potassium phosphate buffer for 5 min at room temperature. The cells were washed three times with H,O for 5 min and then en bloc stained with 2% uranyl acetate (Fisher Scientific Co.) in 25% ethanol for 1 h. Following dehydration through a n ethanol series, the cells were infiltrated gradually with Quetol 651 embedding resin (Ted Pella Inc., Redding, CA) and embedded in a thin layer of resin in LUX petri dishes or in BEEM capsules (Electron Microscopy Sci- ences). After polymerization of the resin, selected cells were